1. Field of the Invention
This invention relates to a quantitative analysis of 3-oxo-5.beta.-steroid by means of an enzymatic method and a reagent therefor.
2. Description of the Background
Among liver function tests those using bile acids have attained an important position in a clinical diagnosis of liver and/or biliary tract diseases in the past several years because of the wide acceptance of the quantitation of the total bile acids in serum employing 3.alpha.-hydroxysteroid-dehydrogenase.
There has been known for long time, as a liver function test, a loading test conducted by measuring the metabolic rate, after administration of dehydrochoric acid which is a typical of 3-oxo-5.beta.-steroid to the body of a subject [J. Japan Internal Medicine, 21, 567 (1933)]. The clinical efficiency of this test can be explained as follows. Namely, dehydrochoric acid administered either orally or otherwise at normal or healthy conditions of a subject is partly transferred to the kidney without being captured at the liver and excreted in urine as it is, but the most of the dehydrochoric acid is captured at the liver, reduced to the reduced form of dehydrochoric acid, and excreted in bile. The reduced form of dehydrochoric acid thus excreted in bile is absorbed at intestine and taken into blood, transferred to kidney, and then excreted into urine. If there are any disorders in the liver function of the subject, only partial reduction of dehydrochoric acid occurs thus giving rise to increased excretion of dehydrochoric acid and decreased excretion of reduced form of dehydrochoric acid in urine as compared to a person with a normal liver function. Likewise, the dehydrochoric acid concentration in blood decreases rapidly if the liver functions with a normal hepatic reduction. The rate of decrease in the blood dehydrochoric acid level lowers as the liver function degrades. The measurement of this function is important in order to determine the reduction-detoxication of the liver.
The quantitative analysis of dehydrochoric acid has been studied since 1930's. The methods of analysis include those employing paper chromatography, thin-layer chromatography, high performance liquid chromatography and the like [J. Yonago Physic, 3, 64 (1951); J. Biochem. 29, 271 (1934)]. The method of tracing labeled dehydrochoric acid has also been proposed [(J. Clin. Invest. 52, 715 (1973)]. These methods are carried out according to the following procedures. When serum is employed as a sample, 1-2 ml of a serum sample is diluted with 9 times by volume of 0.1 N - NaOH physiological saline and passed through a column of Amberlite XAD-2 or the like filler. The column is washed with distilled water and eluted with ethanol to obtain an ethanol solution of bile acids, which is then concentrated by means of an evaporator. The concentrate thus obtained is developed by thin layer chromatography or paper chromatography for separation, using a developing solvent of, for example, butanol/acetic acid/water (10:1:1). The detection limit of this method is approximately 10 .mu.g. When an isotope is employed, a 24-hour urine sample, for instance, is passed through the aforementioned column, extracted with ethanol, concentrated, developed by paper chromatography for separation, and then the quantity of the radio isotope is measured. The detection limit is in the range from 1 .mu.g to 100 .mu.g.
These methods, however, are not employed in a routine assay because of requirements of complicated pretreatment procedures for extraction, concentration, etc. as well as expensive equipment and materials. In particular, the poor measurement sensitivity in these methods poses following problems. That is, urine does not contain the indicative substance of the amount sufficient for the reliable assay. In case of using serum as a sample a large amount of the sample is usually needed because the dehydrochoric acid level in blood is not always sufficiently high after administration of this substance. In addition, the assay may be sometimes hindered by the other components in the serum sample. Determination using an isotope is not necessarily a preferred method because of requirement of special equipment and devices A strong need, therefore, has existed for the development of a convenient and precise quantitative analysis of 3-oxo-5.beta.-steroid.
The inventors have conducted extensive studies for solving the aforementioned problems in the prior art. As a result, it was found that when 3-oxo-5.beta.-steroid.DELTA..sup.4 -dehydrogenase is acted on 3-oxo-5.beta.-steroid in the presence of a reducing chromophoric agent, 3-oxo-5.beta.-steroid is oxidized into 3-oxo-5.beta.-.DELTA..sup.4 -steroid, and at the same time, the reducing chromophoric agent is colored and may be conveniently submitted to a sensitive quantitation. The finding has led to the completion of this invention.